This protocol is used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid. It utilizes a fast high-fidelity non-displacing DNA polymerase ([http://www.neb.com/nebecomm/products/productF-530.asp Phusion]) to replicate the plasmid including the desired mutation. [http://www.neb.com/nebecomm/products/productR0176.asp DpnI] then cuts up all your old plasmid. While this protocol gives more reliable mutations than many other protocols, the constraints may not work for you; check out the [[Site-directed mutagenesis|consensus protocol]] for more options.

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===Procedure===

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==Introduction==

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These protocols are used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid). They both utilize a fast high-fidelity non-displacing DNA polymerase ([http://www.neb.com/nebecomm/products/productF-530.asp Phusion]) to replicate the plasmid including the desired mutation. [http://www.neb.com/nebecomm/products/productR0176.asp DpnI] then cuts up all your old plasmid. While these protocols can make reliable controlled mutations, the constraints may not work for you; check out the [[Site-directed mutagenesis|consensus protocol]] for more options. Also please read both protocols to see which one you want to try.

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==Double Primer Method==

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This protocol uses two oligos, each containing the desired mutation. It is more reliable than the other protocol, but can only mutate at one location (1-3bp) at a time.

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===Method===

1. Design mutagenesis primers.<br>

1. Design mutagenesis primers.<br>

::*The targeted mutation should be included into both primers.

::*The targeted mutation should be included into both primers.

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#Run the following for 20 cycles:

#Run the following for 20 cycles:

##98°C for 10 secs

##98°C for 10 secs

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##60°C for 30 min

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##60°C for 30 secs

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##72°C for 30 sec/kb of plasmid length minimum

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##72°C for 30 sec/kb of plasmid length minimum (this is the TOTAL plasmid length)

This protocol only uses one oligo per mutation site (cuts the oligo cost in half) and can mutate more than one site at a time. While this protocol is less reliable than the previous protocol (only ~25% of colonies will contain the desired mutation(s)), if you're doing multiple mutations on the same piece of DNA than this can save you many days of work by just sequencing a few more colonies. In this protocol there is an initial Kinase (phosphorylation) reaction to allow your primers to be available for ligation. The polymerase will extend from one primer to another and then the Taq ligase will seal the nick. This allows multiple mutations to be done at the same time.

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===Method===

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1. Design mutagenesis primer(s).<br>

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::*The targeted mutation should be in the middle of the primer

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::*Design your primers (including the mutations) to have a [http://www.google.com/search?sourceid=navclient&aq=0&oq=finnzymes+t&ie=UTF-8&rlz=1T4ADRA_enUS407US410&q=finnzymes+tm+calculator Tm] >=78°C.

Current revision

Contents

Introduction

These protocols are used if you want to change one to three consecutive bases in a sequence (like removing a restriction site or changing an amino acid). They both utilize a fast high-fidelity non-displacing DNA polymerase (Phusion) to replicate the plasmid including the desired mutation. DpnI then cuts up all your old plasmid. While these protocols can make reliable controlled mutations, the constraints may not work for you; check out the consensus protocol for more options. Also please read both protocols to see which one you want to try.

Double Primer Method

This protocol uses two oligos, each containing the desired mutation. It is more reliable than the other protocol, but can only mutate at one location (1-3bp) at a time.

Method

1. Design mutagenesis primers.

The targeted mutation should be included into both primers.

The mutation can be as close as 4 bases from the 5-terminus.

The mutation should be at least 8 bases from the 3-terminus.

At least eight non-overlapping bases should be introduced at the 3-end of each primer.

References

Single Primer Method

This protocol only uses one oligo per mutation site (cuts the oligo cost in half) and can mutate more than one site at a time. While this protocol is less reliable than the previous protocol (only ~25% of colonies will contain the desired mutation(s)), if you're doing multiple mutations on the same piece of DNA than this can save you many days of work by just sequencing a few more colonies. In this protocol there is an initial Kinase (phosphorylation) reaction to allow your primers to be available for ligation. The polymerase will extend from one primer to another and then the Taq ligase will seal the nick. This allows multiple mutations to be done at the same time.